Hologram-Based Thermooptical Absorbance Detection in Capillary Electrophoresis: Separation of Nucleosides and Nucleotides

Abstract

On-column thermooptical absorbance (TOA) detection in capillary electrophoretic separations of various nucleoside and mono-and diphosphate nucleotide mixtures absorbing at 257 nm is demonstrated in 20 fim i.d. capillaries. The analytes are optically pumped by a frequency-doubled argon ion laser and probed by a laser diode or by a He/ Ne laser beam guided to the detection volume by a holographic optical element Absorption detection limits of 2.2 //AU using time constants of 0.3 s and 20 mW of UV power are obtained over a linear dynamic range covering three to four decades. As higher pumping power is required to enhance the thermooptical sensitivity, photobleaching appears as a major problem in the quest for lower detection limits for some of the substances studied such as deoxyuridine and uridine. Concentration detection limits as low as 50 nM for adenosine monophosphate, corresponding to a mass detection limit of 0.4 finol, and separation efficiencies up to 320 000 theoretical plates are measured. A theoretical model, which translates the obtained TOA signals into absorbances, is proposed and describes the TOA effect for smaller capillaries rather well.Various on-column optical detection methods have been devised for capillary electrophoresis (CE). When the sample of interest contains a fluorescent chromophore that coincides with a lasing line, laser-induced fluorescence (UF) is undoubtedly the best choice. Using this method, concentration detection limits (LODs) as low as 10"10 M (0.2 am injected) for native bovine serum albumin (BSA) have been reported.* 1 However, as the number of naturally fluorescent samples is small, and the laser emission wavelengths are limited, this case should be considered as an exception rather than the rule.Concentration LODs in the 10-11 M range, or lower, are obtained in CE using precolumn labeling with fluorescent tags and LIF detection. However, direct derivatizations (samples are first derivatized at high concentrations and then diluted to the impressive low levels reported) below the 10"' M range have not been reported to date. Furthermore, derivatization procedures are not always available for the substance of interest or are often rather tedious and, when available, often lead to quantification problems because the extent of derivatization is not always

Sensitive photothermal interferometric detection method for characterization of transparent plate samples

“…In recent years, due to the rapid developments of HPLC and CE separation techniques, a number of interference methods based on transmitted-beam interference [33][34][35] have been developed and successfully used as universal refractive index ͑RI͒ detection for HPLC and CE separations. Keeping other conditions the same, the retroreflected beam interference method is potentially more sensitive than the transmitted beam interference approach because the probe beam passes twice through the detection region in the retroreflected beam configuration.…”

Analyses of anticancer drugs by capillary electrophoresis: a review

“…Methylated purine, pyrimidine and other modified nucleosides have been shown to be excreted in abnormal amounts in urine of patients with cancer (Watanabe and Maekawa, 2010;Hatziapostolou and Iliopoulos, 2011). Elevated concentrations have been suggested as possible markers of different forms and stages cancers (Hsu et al, 2011;Vareed et al, 2011;Markuszewski et al, 2010;Struck et al, 2011;Krattiger et al, 1995;Masuda et al, 1993) It was concluded that the levels of nucleosides in urine from cancer patients were, generally, elevated, and that the increase in modified nucleosides was more pronounced than that of normal nucleosides. In addition, the concentrations of pseudouridine (pseu), 1-methylinosine (m1I), N4-acetylcytidine (ac4C), 1-methylguanosine (m1G) and 2-methyl guanosine (m2G) in urine of cancer patients were elevated significantly.…”

Capillary electrophoresis‐electrospray‐mass spectrometry of nucleosides and nucleotides: Application to phosphorylation studies of anti‐human immunodeficiency virus nucleosides in a human hepatoma cell line

“…Micellar electrokinetic capillary chromatography (MEKC) has also been used for the determination of azidothymidine triphosphate [17] and nucleosides [8]. On-column thermo-optical absorbance detection has also been demonstrated in capillary electrophoretic separations of various nucleosides and nucleotides mixtures in MEKC modes [18]. A dynamic pH junction in CE for improved focusing of nucleotides has been introduced for improved concentration sensitivity [19].…”